Covalent histone modifications are considered one of the most important epigenetic phenomena. Histone demethylases are the most recently discovered class of histone-modifying enzymes. Over a dozen Jumonji C domain-containing histone demethylases (JHDMs) have been characterized so far. They can modify lysine residues at many different positions on histone proteins at all methylation states. They often display tissue-specific expression and play critical roles in a variety of cellular processes, such as gene expression, meiosis, and embryonic stem cell self-renewal. In addition, several JHDMs such as JMJD2C have been associated with human diseases, such as mental retardation and cancers. However, the cellular functions and the substrate scope of JHDMs are still unclear. Currently, research tools to study JHDMs and their cellular functions are very limited. Highly active and specific chemical probes for JHDMs currently do not exist. A uniform biochemical assay for various JHDM isoforms is also underdeveloped. In this proposal, we plan to develop a series of specific chemical probes for JHDMs, including a highly active and specific cellular probe, a fluorescent small-molecule probe, and a highly functionalized peptidic probe specifically targeting H3K9-JHDMs. The cellular probe can be used for the studies of the cellular functions of JHDMs and histone methylation dynamics in a wide range of biological processes and disease processes. The fluorescent probe is being applied to develop a fluorescence polarization-based assay, which will not only enable us to validate our bivalent inhibitor design, but also provide a uniform JHDM biochemical assay to analyze isoform specificity of JHDM inhibitors. The peptidic probe is important not only to study the substrate scope of JHDMs, but also to provide valuable information on the histone crosstalk. In addition, the strategy we develop here can be applied to the discovery of novel JHDMs that modify H3K79me3 and H4K20me3, eventually leading to a systematic map of histone methylations and their corresponding JHDMs. Furthermore, these probes can also be used to validate JHDMs as potential novel therapeutic targets for diseases such as cancers, and can serve as lead compounds for further development of novel therapeutics for these diseases.